Abstract: Ciliopathies are an expanding group of human disorders whose cellular basis can be traced to defects in the formation or function of small, cellular organelles called cilia. Cilia are found on almost every cell type in the vertebrate body and thus ciliopathies can encompass defects in multiple organs and tissues. Strikingly, ciliopathies span a spectrum, ranging from infertility to neonatal lethality but the genetics underlying these divergent phenotypes are poorly understood. Defining the genes and genetic interactions that regulate cilia function is essential to understanding the etiology of these diseases as well as for developing future therapies. We have developed mice carrying a spontaneous mutation (hop) in the Ift56 gene into a new model for defining the genetic control of ciliopathy severity. Ift56 is a highly conserved cilia-localized protein that is required for cilia function in several model organisms. We recently discovered that while the Ift56hop phenotype is viable and mild on the Balb mouse background, it is lethal on the B6 background. The phenotype of the hop-B6 mutant models VACTERL Association in humans and overlaps with the Ift27 mutant ciliopathy phenotype. Our objectives are to 1) utilize the hop-B6 mutant as a means to understand VACTERL disorders, 2) define the function of Ift56 in mammalian cilia, and 3) isolate genetic loci that interact with the Ift56 mutation in order to elucidate the genetic landscape underlying ciliopathies. Our First Aim focuses on analyzing the tissue defects in Ift56-B6 mutants. Histological approaches coupled with gene expression analysis and microCT imaging will provide key insight into signaling and developmental patterning events controlled by Ift56 and the VACTERL-associated defects in hop-B6 mutants Aim2 explores the ciliary roles of Ift56, and how these are altered in the B6 and Balb backgrounds. Using candidate and proteomic approaches, we will determine the set of proteins that require Ift56 function for their localization and trafficking within cilia. Finally, we test the requirement of Ift56 for IFTB complex integrity. In our Third Aim we map, isolate and validate modifiers of the hop phenotype in the B6 and Balb backgrounds. We will also examine genetic interactions between Ift27 and hop mutants as well as the effects of the Balb modifier background on the Ift27 phenotype. These experiments will identify key genetic modifiers of ciliopathy severity and provide new targets for resequencing in ciliopathy patients. Together, the proposed studies will uncover the conserved functions of Ift56, a key cilia protein, by defining how Ift56 regulates cilia and organogenesis. More broadly, these studies have the opportunity to uncover unique insights into how genetic variants across the genome modify ciliopathy severity. Results from this study will shed key and novel insights into cilia biology and ciliopathies, and lay the groundwork for future diagnostic and therapeutic strategies.